In recent years, a treatment in which cells are collected from body fluid or tissue of a patient or a donor, cultured to be amplified and processed, and transplanted to an affected area, so-called, regenerative medicine and cell medicine have been drawing attention. In the regenerative medicine and cell medicine, in many cases, useful cells to be used for a treatment are separated from a cell group collected from body fluid or tissue, and then the cells are amplified by culture. The cells obtained by culture are transplanted to, for example, a skin, a bone, a cartilage, or a cornea and are established to be safe and effective in some diseases of these organs. Therefore, such a medicine is expected to be generalized as a beneficial treatment method for patients (for example, see Non-Patent Document 1).
In recent years, it has been revealed that bone marrow fluid, cord blood, and others include adherent adult stem cells having characteristics of capable of being differentiated into various cells, such as bone cells, cartilage cells, muscle cells, and adipose cells (for example, see Non-Patent Documents 2 to 4). The adult stem cells can be differentiated into a wide variety of cells and organs. On this account, a method of efficiently separating and amplifying the adult stem cells is very important from a viewpoint of the development of regenerative medicine. At present, the adult stem cells are typically separated by a density gradient centrifugation method such as Ficoll-Paque fractionation (for example, see Non-Patent Document 5). However, the method requires a complicated procedure in which cells are repeatedly washed using a centrifuge for separating cells from a separation liquid. In addition, the method is accompanied by the risk of damage to cells due to the centrifugation or of contamination due to operation in an open system.
It was reported that the separated adult stem cells are present at a very small frequency of one in 104 to 106 nucleated cells in adult bone marrow fluid (for example, see Non-Patent Document 5). Hence, the separated adult stem cells are required to be cultured and amplified until the number of cells reaches a required value for treatment. At present, adult stem cells are typically amplified by culture using, for example, a petri dish, a flask, a culture bag, or a cartridge for culture in a CO2 incubator at a temperature of 37° C. In this case, a culture medium exchange operation and a passage operation are carried out by an operator. Thus, each operation is accompanied with the risk of contamination and takes some time.
On this account, in recent years, automatic culture devices that automatically carry out culture operations such as seeding of cells and exchange of a culture medium and do not require labor have been developed (for example, see Patent Documents 1 to 3). However, current automatic culture devices have a single function of automatically culturing desired cells and do not have a function of separating desired cells from body fluid or tissue.
After amplifying intended cells until the number of cells reaches a predetermined value, the cells are treated with an enzyme such as trypsin or with a divalent cation chelating agent to be detached from a culture container and the detached cells are collected together with a liquid containing such an enzyme. Therefore, it is required to separately carry out an operation for removing the enzyme such as trypsin or the divalent cation chelating agent.
At present, a most common method of washing cells is centrifugation. In the method, cells are precipitated by centrifugation, a supernatant is removed, then a new washing liquid is added, the cells are re-suspended, and centrifugation is carried out once again. Such a procedure is repeated several times, thereby washing cells. However, for example, the procedure is complicated and a separate operation is not easy in a closed system. Therefore, the cell washing process is noted to involve the risk of contamination.
There is a method in which cultured cells are trapped using a separation material such as nonwoven fabric and the trapped cells are washed and then collected (for example, see Patent Document 4). However as with the centrifugation method, such a technique is used alone. Separation, culture, and collection of particular cells cannot be performed by a single device.